News for Bacteria Monitors

Monitoring Best Practice Reminders

On this page, you will find bacteria monitoring best practices. To go to the step-by-step protocol, please click the blue button:

Review Bacteria Monitoring Protocol

• Keep your sample on ice. After collecting your sample, store it in a Ziploc bag with some ice cubes or an ice pack. When you return home, store it in the refrigerator until you're ready to plate it. Don't store your sample in the freezer.
• Shake it up. Shake your sample bottle 25 times before opening the bottle and pipetting a quantity into the growth media bottle.
• Keep it warm, but not that warm. The ideal incubation temperature is 35 C (95 F). However, you can incubate at 30-40 C (86-104 F) with no problem. If the temperature goes above 45 C or so, general coliforms will not grow at temperatures above 40 C.
• Overwhelmed by teals? Are teal colonies ritually filling up your plate? Per Virginia Department of Environmental Quality (DEQ), 38 C for incubation isn't too hot for E. coli and it might help discourage teal growth. Don't go nuts, though! Temperatures above 40 C are too high.
• If your incubator runs hot, position the Petri dish so that it's on the opposite side from the bulb so that it doesn't get too hot. You can also take out the aluminum foil to lower temperatures. Your incubator may run hotter in the summer. 
• Getting it just right. See the image to the right for a recommendation of how to keep your plates at the proper temperature. Notice that the dome hangs off the edge a bit, and the plates are on the opposite side from the light bulb and aren't over top the light bulb.
• If your incubator runs cool, try adding more aluminum foil. You can also ask the program coordinator for a new light bulb. Your incubator may run cooler in the winter. 
• Swirl it for 10 seconds. Swirl or roll the bottle with the media and water sample for 10 seconds before pouring it into the Petri dish. Do not shake it.
• Not gelling? If a portion or all of the plate doesn't gel, this is likely the fault of a "bad" Petri dish that wasn't coated properly. If this happens, try again using some of your remaining sample water and "fresh" media and Petri dishes.
• Save the leftovers. When you plate your sample, save your "left over" water sample in the fridge until after you're done and have read the plate. If something goes wrong or is questionable, you'll need the sample to run another test.
• Extra moisture on top of the media before you incubate? If there's some moisture on your plate after 90 minutes of setting, go ahead and turn the plate over and put it in the incubator. If your sample fails in some way, do a second try with some of the "left over" water.

When Plates Do Not Properly Incubate

There will likely be times when you will need to re-plate your sample because something went wrong during incubation. Each instance below requires the water sample to be re-plated.  

Extremely heavy bacteria growth. E. coli are still countable.

"Holes." Note that you can see the floor's pattern through the hole in the gel.

Haze. There are several potential causes for haze including:

• Not flipping over the petri dish during incubation and condensation impacted the result
• Using a lot of sample water (5ml), not mixing it thoroughly enough, and condensation impacted the result
• The petri dish is bumped or disturbed while solidifying
• The media has thawed, but is still really, really cold when mixed with the sample water, resulting in a lot of condensation
• The petri dishes are coated with a substance that reacts with the media. The interaction of these causes the gel to form. If the spray was not evenly applied, when the petri dish was flipped over, the gel pulled a little away from the petri dish, bacteria got in this “middle” space between the dish and the gel, and some bacteria grew in this space.

Holes. The media interacts with a coating that was sprayed onto the petri dish bottom to form the fel. If a section of the dish was missed by the spray, a "hole" will occur in that location.

Overwhelmed by Teals. If your plate has a significant number of teal colonies, and they are located in close proximity to other blue colonies, it can be very difficult to read the plate. Do the best that you can, read the plate, and record your findings. Start a new plate from the remaining water sample, incubate at 38 degrees Celcius, and record that information on the Google doc, too.

Incubation Temperature is Too High. Sometimes the incubators may get cooking higher than we'd like. If you find that your incubator is cooking at more than 40 degrees Celcius, and it has only been a couple hours, adjust the incubator and bring the temperature down. If you do not know how long the incubator was cooking at this high temperature and it may or may not have been going on for an extended period of time, record that plate's data and start another plate from the same water sample.

Counting Colonies

"Pinpoint" colonies, extremely small colonies that are a little larger than the dot of an "i," are not counted if they make up a small percentage of the total number of colonies on the plate. These very small pinpoint colonies may be the result of a bacterium with damaged DNA, a different type of bacteria (not E. coli) may be starting to grow on the plate, or there was a lack of adequate food to support this particular bacterium's growth. See the Virginia Department of Environmental Quality guide to help you identify the bacteria colonies.

Quality Assurance Project Plan (QAPP)

A QAPP was initially approved by VA DEQ for Arlington's bacteria monitoring program in March 2013. The QAPP was updated August 2014. All of our monitoring protocols are based on the information in this document.  

Salinity Testing Instructions 

From December - February we also measure salinity on our regular monitoring dates and on storm dates. 

(PDF, 407KB)Salinity Testing Instructions(PDF, 410KB)